Derivatives of the cyclopentanoperhydrophenanthrene series and process of making same



Patented Apr. 2, 1946 DERIVATIVES OF THE CYCLOPENTANO-PERHYDROPHENANTHRENE SERIES AND PROCESS OF MAKING SAME 'Thomas F.Gallagher and William P. Long, Chicago, Ill., assignors to theGovernment of the United States of America, as represented by theSecretary of War and his successors in ofiice No Drawing. Application June 19, 1944, Serial No. 541,096

4 Claims.

l'his invention relates to hormone-like compounds of the corticalsteroltype, particularly hormone-like derivatives ofcyclopentanoperhydrophenanthrene having an oxygen function at carbon I Iand to an improved method of synthesizing these compounds.

It has been known for some time that the adrenal steroids were importantmedical compounds having particular utility in hormone therapy. Thesecompounds are characterized by their complex molecular structure andhave the following ring system: I I

The various processes of preparing these compounds suggested heretoforehave been relatively unsatisfactory; Thus, the prior processes have beenfound tedious, complicated and difiicult to control and the resultingyields therefrom of desired sterols too low for practical operation.

This invention has as its object a practical and economical process ofpreparing hormone-like cortical steroid type compounds. Another objectof this invention is to provide a method of preparing a hormone-likederivative of cyclopentanoperhydrophenanthrene having an oxygen functionat carbon I I.

'Other objects will be apparent from the following description oftheinvention.

The'compoundsof the present invention have an oxygen function, e. g. ahydroxyl or keto or substituted hydroxyl or keto group at the carbon llposition of the steroid nucleus. In carryin out the invention, thecompounds are prepared from cyclopentanoperhydrophenanthrenederivatives, l. e. compounds with asteroid nucleus characterized by aketonic group at the carbon 12 position of the nucleus. Previousattempts to introduce an oxygen function at carbon 11 and still maintaina reactive keto group at carbon 12 have been unsuccessful. In priorprocesses, the introduction of an oxygen function at the carbon 11position of the sterol nucleus has resulted in the formation ofrearrangement products lacking an active keto group at the carbon 12positiongroup without destroying the oxygen function introduced at thecarbon 11 position.

above about 40 C., and preferably at roomtempera-ture (20 C.), ofasteroid nucleus characterized by a halogen group at the carbon 11position and a ketonic group at the carbon 12 position. The temperatureat which the hydrolysis is carried out is of utmost importance since ithas been found that substantially no-sterol compound having an oxygenfunction at the carbon 11 position and a reactive keto group at thecarbon 12 position is formed by hydrolysis at temperatures above 40 C. Apreferred illustrative method of practicing the present invention is asfollows:

1. Preparing an alkyl desoxycholate by esterifying commercialdesoxycholic acid;

2. Preparing a 3-acyl derivative of the alkyl desoxycholate V by partialacylation of the ester product obtained by step one; u

3. Preparing a. 12-keto derivative of the 3-acylalkyl desoxycholate byoxidizing the product ob tained by step two;

4. Preparing an ll-brom derivative of the 3- acyl-12-keto alkyldesoxycholate by brominating the product obtained by step three;

5. Preparing 3-11-dihydroxy-12-keto cholanic acid by hydrolysis at atemperature not above about 40 C. of the ll-bromproduct obtained by stepfour.

The desired product or epimers having a reactive keto group at carbon 12may be separated from undesired products by fractional crystallizationor by various other means known in the it art. One preferred method ofseparation is based upon the discovery that substantially all of thedesired product occurs as an epimer which forms an insoluble sodium saltwhile the undesired products for the most part form soluble sodiumsalts. The insoluble sodium salt may be isolated and treated as follows:

6. Preparing the hydrazone of the insoluble sodiumsalt of3,11-dihydroxy-12 keto-cholanic acid obtained by step five;

7. Reduction of the hydrazone derivative ob tained by step six wherebythe hydrazone group which replaced the keto group is converted to amethylene group at the carbon 12 position of the nucleus;

8. Isolation of the 3,11-dihydroxy cholanic acid obtained by step sevenas an alkyl ester.

The following example serves to illustrate the present invention but isnot intended to limit it thereto:

I. Methyl Desoxycholate: Commercial desoxycholic acid [3(a) 1'2dihydroxy'cholanic acid] is first esterified by suspending 450 grams ofwell dried desoxycholic acid in approximately one liter of dry methanol,adding 0.5 to 1.0 cc. of concentrated sulphuric acid in 25 cc. ofmethanol and, after stoppering the flask, allowing the mixture to standat room temperature for twenty-four hours. Over this period thedesoxycholic acid dissolves and the methyl ester crystallizes. Thereaction mixture is then filtered with suction, the precipitate Washedwell by suspension in very cold methanol and dried. The materialobtained melts at 97-102 C. (corrected) and, although somewhat impure,is satisfactory for use in the further steps of the process.

II. 3(a) acetoxy 12 hydroxy' methyl cholanate: 400 gramsof methyldesoxycholate .obtained by step I are dissolved in 750 cc. of pyridinewhich has been distilled over barium oxide. To this solution, 165 cc.(1.6 mole) of freshly distilled acetic anhydride are added and themixture alwith water andall aqueous 'fractionsjdiscarded.

The semi-crystalline residue obtained during the reaction is thendissolved in the ether and the resulting solution extracted with smallportions I dition to the water is too fast the product is oily andwashing thereof difficult. The product obtained is filtered with gentlesuction and washed five times by suspension in water and vigorousmechanical stirring. The filtrates are yellow and are discarded. Afterthe last washing, the product which is a pale yellow to white powderysolid is filtered as dry as possible and dissolved in six liters ofninety-five per cent ethanol. a

V. 3,11-dihydroxy-12-keto-cholanic acid: To the alcohol solution of thebrom-ketones obtained by step-IV, four liters of 2.6 N sodium hyof fivepercent sulphuric acid-five per cent sodium carbonate and finallythoroughly with water. After drying over sodium sulfate, the ether isdistilled off in the usual manner. The crystalline residue obtained maybe recrystallized from methanol and melts at about -124-126 C.

III. 3(a) acetoxy '12-keto methyl cholanatez 'ture is allowed to standwith continued stirring for another hour and then about 15'00 cc.'-ofwater are added slowly whilexstirring is com tinued. The crystallinemass is filtered, washed twice by suspension in water, filtered as dryas, possible and dissolved in ethanol. This requires a large volume ofalcohol to effect solution and since the product crystallizes with greatease, the solution must be filtered while very hot. The crystallineproduct obtained, 3(a) .acetoxy l2- keto methyl cholanate, melts atabout 147% 14.9" C. and is sufficiently pure for bromination purposes. I

IV. 3(a) acetoxy 11 brom-l'2-keto methyl cholanate: About 410 grams ofthe keto ester obtained by step III are dissolved in one liter of stableacetic acid and 295 cc. of 6.8 'N Brz in glacial acetic acid added (1.1'molesBrz). The mixture is stoppered and set aside at room temperaturefor "about five days. The reaction solution is next poured slowly from adroppingv funnel into a large quantity of ice cold water'with continuedvigorous stirring. If the addition is made slowly and the stirring "iscontinuous the product'is sandy and easily filtrable. If the addroxideare added with stirring, the bottle stopperedand set aside at roomtemperature for about twenty-four to thirty hours. It is important thatthehydrolysis be conducted at low temperature, i. e. not above about 40C. If the reaction mixture is heated, the resulting products do notcontain (or retain)v the active keto group at carbon 12. After standing,a heavy' precipitate forms; the solution is next chilled overnight at-10 C. and filtered withvgentle suction while still cold. Theprecipitate is washed twice by suspension in a small volume of ice coldwater and dried without heating.v

The precipitate thus obtained, contains. a desired (reactive keto)epimer as an insoluble sodium salt while the filtrate retains for themost part the undesired products as soluble sodium salts. One epimerwith a reactive '12-keto group does form a soluble sodium salt. It mayberecovered from the filtrate by crystallization if desired, although asit is present only in small amounts its recovery has generally not beenfound worthwhile. This diiierence in solubilities of the sodium saltsprovides (an unusually satisfactory means for separation and recovery ofsubstantially all of the desired product characterized by the reactiveketo group.

Additional insoluble sodium salt maybe recovered from the filtrate orreaction mixture by acidifying; the filtrate under -ether, extractingwith ether, washing the extract with water, drying the washed extractover sodium sulfate, :distilling on the ether, dissolving the oilyresidue in ethanol; neutralizing with caustic alkali, diluting withWater and allowing the product to stand at about '10 C. .The insolublesodium salt obtained as a precipitate'is then washed by suspension incold water and dried without heating.

VI. Hydrazone of the insoluble sodium salt of 3,11-dihydroxy-12-ketocholanic'acidi About 100 grams of the dried insoluble sodium saltobtained by step V are dissolved in 1400 cc. of absolute ethanol underreflux. To the resulting solution 25, cc. of e 100 per cent hydrazinehydrate are added and refluxing continued for one hour. Approximatelytwo-thirds of the alcohol is then distilled off and the residualsolution allowed to crystallize. The mother'liquors from this crop ofcrystals upon further concentration yield several additional cropsofhydra zone. The hydrazone obtained is quite hygroscopic and should bestoredina dessicator. p

VII. Wolff-Kishner reduction of the 3. -dihydroxy-12- keto cholanic acid.hydrazone: About 5 grams of the hydrazone obtained by step VI areintroduced into 'a glass or steel bomb tube and cc. of sodium ethylateprepared from 3 grams of sodium added along-with 0.5 .cc. of onehundredper cent hydrazine hydrate. The tube is then cooling, the tube is opened.and the contents rinsed outwith alcohol, water or other solvent.

VIII. Isolation of 3,1l-dihydroxy cholanic acid as the methyl ester: Thereaction product obtained by step VII is preferably rinsed from the bombtube with ninety-five per cent ethanol so that the final volume is oneliter of ethanol for each 40 grams of the sodium salt hydrazone reduoed.This solution is heated and neutralized to phenolphthalein with diluteaqueous hydrochloric acid and an additional amount of water sufiicientto bring the volume to two liters added. One liter of 0.3 N bariumhydroxide is next added, to the hot aqueous alcohol solution and finallyone liter of hot water added slowly with constant stirring. The flask isthen stoppcred and allowed to cool overnight. The insoluble barium saltis removed by filtration and the filtrate containing soluble bariumsalts acidified under ether and extracted twice with fresh portions ofether. The ether solution is washed thoroughly with water,

HO 041150 OOH 1 Esterlfication (MeOH) 1 HO CAHaCOOMe i Acetic aiihydrlde(Pyridine) HO 014E800 OMe AcO OrOa

III 0 1 Br: 3i:

C HgCOOMe AcO Alkali Hydrolysis (NaOH) AcO evaporated to dryness, andtraces of water removed from the residue by distilling benzene two orthree times from the residue. (The free acid may be isolated from thisfraction by acidification with excess hydrochloric acid and filtration.)The oily material is preferably dissolved in dry methanol, a smallamount of concentrated sulphuric acid added, and the solution allowed tostand at room temperature for from four to five 0 cimoopiva l ydrazineHzNz C HaO 0 ONa reduction CAHSCO ONa VII Reacidificetion ZCitCOOH v UEsterlflcation (MeOH) VIII CAHlCOOMB 7 Having thus described ourinvention, what we about 40 C. to form an alkali salt of3,11-dihydroxy-l2-keto cholanic acid reacting said salt with hydrazineto form the 3,11-dihydrxy-12- hydrazone derivative thereoi andthereafter reducing said l2-hydrazone derivative to form 3,11-clihydroxycho lanic acid.

2. The compound"3,l'l-dihydroxy cholariicacid. 3. A process for theproduction of 3,11-dih drox 'ch olanic acid which comprises subjectingalkali hydrolysis at a'tempera'ture not above about 40 G. to form analkali salt of 3,ll'-'dihy'dioily+ 12-k'et6 choianic aciti, reacting-sawsalt with hydrazine to form the 3,11-dihylroiZy-l'2-liydrazonederivative thereof and thereafter reducing said 7 12-hydrazonederivative to form 3,=1 '1 olihydroi y cholanic aci'gi.

4. A process for the production of 3,l-l-dihydroxy chol'anio acidwhiohoomprises hydroiyzing 3(u)-' acetoxy-l1-brom-12-keto 'me't'h'ylcholonate with sodium hydroxide at'a temperature of about C. to form aninsoluble sodium s'alt of 3,11'-dihyciroxy-lZ-keto cholar'ilc acid,reacting said salt with hydrazine to term the 3,1'1-dih'ydroxy-12-hydra'zo'ne derivative thereof, and thereafter re-' ducing said12hydrazoiie'derivative to form 3,11- uiiiydroxy cholan'ic acid. 7 I

THOMAS E. GALLAGHER. WILLIAM P. LONG.

